Our long-term objectives are to define the pathway by which two cells fuse to become one. Fertilization is a fundamental process common to all sexually reproducing organisms, but cell fusion also occurs during development. We propose to continue analysis of genes required for two major steps in yeast mating, cell fusion and nuclear envelope fusion. Many genes required for cell and nuclear fusion have homologs in all eukaryotic organisms and their study will provide important clues to human cell biology and disease. During development, cells go from proliferation, devoted to cell division and lacking specialized functions, to differentiation, in which cell division ceases and specialized cell functions are expressed. Proliferation and differentiation are mutually exclusive; during development cells shut down mitosis as they turn on specialized cell functions. One hallmark of cancer cells is the loss of differentiated functions as cells re-acquire the capacity for unrestrained proliferation. Thus coordination of mitosis and differentiation is of vital importance Similarly, yeast cells exit the cell cycle and express specialized proteins as they differentiate ito mating cells. Gene expression begins before the completion of the previous cell cycle; cells must also prevent premature activation of mating. One way yeast cells block premature mating is by retaining a key activator of cell fusion, Fus2p, in the nucleus until mitosis is complete. We propose to determine how yeast cells couple nuclear retention to the cell cycle, focusing on a critical protein kinase, Cla4p, and a phosphatase, which together regulate Fus2p localization. We will next determine how cells reenter the mitotic pathway by targeting the degradation of the proteins induced during mating. We will also determine how a transcriptional activator of mating, Kar4p, is able to support a different regulatory program during entry into meiosis. Although numerous cell fusion events occur during development and disease, the mechanism of cell fusion is poorly understood and few cellular fusogens have been identified. We propose to use high-throughput imaging as part of a genomic screen to identify the cellular fusogen required for yeast mating. We will also use genetic and biochemical approaches to identify the downstream effectors of cell fusion, activated by Fus2p and Cdc42p, the highly conserved regulator of actin and cell polarity. We will use advanced imaging of membrane trafficking to identify the signaling mechanism(s) that regulate commitment and progression into cell fusion. At the culmination of mating, the nuclear envelopes fuse to form a single diploid nucleus. The nuclear envelope has two membranes, requiring two coordinated fusion events. Fusion of the outer membrane is related to ER fusion and mutations in this pathway have been identified in hereditary spastic paraplegia. How the inner membranes fuse is not known. We hypothesize that Kar5p, a conserved mating-induced protein, couples the inner and outer nuclear membranes during fusion and facilitates inner-membrane fusion. We will use advanced imaging and genetic methods to identify Kar5p's role(s) in nuclear membrane fusion.